A low concentration of ouabain (0.18 µg/kg) enhances hypertension in spontaneously hypertensive rats by inhibiting the Na+ pump and activating the renin-angiotensin system

We investigated the effects of low ouabain concentrations on systolic (SAP) and diastolic (DAP) arterial pressures and on pressor reactivity in 3-month-old male spontaneously hypertensive rats (SHR). Arterial blood pressure (BP) and pressor reactivity to phenylephrine (PHE) were investigated before and after 0.18 μg/kg ouabain administration (N = 6). The influence of hexamethonium (N = 6), canrenone (N = 6), enalapril (N = 6), and losartan (N = 6) on ouabain actions was evaluated. Ouabain increased BP (SAP: 137 ± 5.1 to 150 ± 4.7; DAP: 93.7 ± 7.7 to 116 ± 3.5 mmHg; P < 0.05) but did not change PHE pressor reactivity. Hexamethonium reduced basal BP in control but not in ouabain-treated rats. However, hexamethonium + ouabain increased DAP sensitivity to PHE. Canrenone did not affect basal BP but blocked ouabain effects on SAP. However, after canrenone + ouabain administration, DAP pressor reactivity to PHE still increased. Enalapril and losartan reduced BP and abolished SAP and DAP responses to ouabain. Enalapril + ouabain reduced DAP reactivity to PHE, while losartan + ouabain reduced SAP and DAP reactivity to PHE. In conclusion, a small dose of ouabain administered to SHR increased BP without altering PHE pressor reactivity. Although the renin-angiotensin system (RAS), Na+ pump and autonomic reflexes are involved in the effects of ouabain on PHE reactivity, central mechanisms might blunt the actions of ouabain on PHE pressor reactivity. The effect of ouabain on SAP seems to depend on the inhibition of both Na+ pump and RAS, whereas the effect on DAP seems to depend only on RAS.

Effects of small doses of ouabain on the arterial blood pressure of anesthetized hypertensive and normotensive rats

Ouabain increases vascular resistance and may induce hypertension by inhibiting the Na+ pump. The effects of 0.18 and 18 µg/kg, and 1.8 mg/kg ouabain pretreatment on the phenylephrine (PHE; 0.1, 0.25 and 0.5 µg, in bolus)-evoked pressor responses were investigated using anesthetized normotensive (control and uninephrectomized) and hypertensive (1K1C and DOCA-salt treated) rats. Treatment with 18 µg/kg ouabain increased systolic and diastolic blood pressure in all groups studied. However, the magnitude of this increase was larger for the hypertensive 1K1C and DOCA-salt rats than for normotensive animals, while the pressor effect of 0.18 µg/kg ouabain was greater only in DOCA-salt rats. A very large dose (1.8 mg/kg) produced toxic effects on the normotensive control but not on uninephrectomized or 1K1C rats. Rat tail vascular beds were perfused to analyze the effects of 10 nM ouabain on the pressor response to PHE. In all animals, 10 nM ouabain increased the PHE pressor response, but this increase was larger in hypertensive DOCA-salt rats than in normotensive and 1K1C rats. Results suggested that a) increases in diastolic blood pressure induced by 18 µg/kg ouabain were larger in hypertensive than normotensive rats; b) in DOCA-salt rats, smaller ouabain doses had a stronger effect than in other groups; c) hypertensive and uninephrectomized rats were less sensitive to toxic doses of ouabain, and d) after treatment with 10 nM ouabain isolated tail vascular beds from DOCA-salt rats were more sensitive to the pressor effect of PHE than those from normotensive and 1K1C hypertensive rats. These data suggest that very small doses of ouabain, which might produce nanomolar plasma concentrations, enhance pressor reactivity in DOCA-salt hypertensive rats, supporting the idea that endogenous ouabain may contribute to the increase and maintenance of vascular tone in hypertension.

Effects of ouabain on vascular reactivity

Ouabain is an endogenous substance occurring in the plasma in the nanomolar range, that has been proposed to increase vascular resistance and induce hypertension. This substance acts on the a-subunit of Na+,K+-ATPase inhibiting the Na+-pump activity. In the vascular smooth muscle this effect leads to intracellular Na+ accumulation that reduces the activity of the Na+/Ca2+ exchanger and to an increased vascular tone. It was also suggested that circulating ouabain, even in the nanomolar range, sensitizes the vascular smooth muscle to vasopressor substances. We tested the latter hypothesis by studying the effects of ouabain in the micromolar and nanomolar range on phenylephrine (PE)-evoked pressor responses. The experiments were performed in normotensive and hypertensive rats in vivo, under anesthesia, and in perfused rat tail vascular beds. The results showed that ouabain pretreatment increased the vasopressor responses to PE in vitro and in vivo. This sensitization after ouabain treatment was also observed in hypertensive animals which presented an enhanced vasopressor response to PE in comparison to normotensive animals. It is suggested that ouabain at nanomolar concentrations can sensitize vascular smooth muscle to vasopressor stimuli possibly contributing to increased tone in hypertension.

Relationships among endogenous ouabain, alpha-adducin polymorphisms and renal sodium handling in primary hypertension.

OBJECTIVE: The basolateral Na pump drives renotubular reabsorption. In cultured renal cells, mutant adducins, as well as sub-nanomolar ouabain concentrations, stimulate the Na-K pump. METHODS: To determine whether these factors interact and affect Na handling and blood pressure (BP) in vivo, we studied 155 untreated hypertensive patients subdivided on the basis of their plasma endogenous ouabain or alpha-adducin genotype (ADD1 Gly460Trp-rs4961). RESULTS: Under basal conditions, proximal tubular reabsorption and plasma Na were higher in patients with mutated Trp ADD1 or increased endogenous ouabain (P = 0.002 and 0.05, respectively). BPs were higher in the high plasma endogenous ouabain group (P = 0.001). Following volume loading, the increment in BP (7.73 vs. 4.81 mmHg) and the slopes of the relationship between BP and Na excretion were greater [0.017 +/- 0.002 vs. 0.009 +/- 0.003 mmHg/(muEq min)] in ADD1 Trp vs. ADD1 Gly carriers (P &lt; 0.05). BP changes were similar, whereas the slopes of the relationship between BP and Na excretion were lower [0.016 +/- 0.003 vs. 0.008 +/- 0.002 mmHg/(muEq min)] in patients with low vs. high endogenous ouabain (P &lt; 0.05). In patients with high endogenous ouabain, volume loading increased the BP in the ADD1 Trp group but not in the Gly group (P &lt; 0.05). Thus, patients with ADD1 Trp alleles are sensitive to salt and tubular Na reabsorption remains elevated after volume expansion. CONCLUSION: With saline loading, BP changes are similar in high and low endogenous ouabain patients, whereas tubular Na reabsorption increases in the high endogenous ouabain group. Saline loading unmasks differences in renal Na handling in patients with mutant adducin or high endogenous ouabain and exposes an interaction of endogenous ouabain and Trp alleles on BP.

Effects of ouabain on vascular reactivity

Ouabain is an endogenous substance occurring in the plasma in the nanomolar range, that has been proposed to increase vascular resistance and induce hypertension. This substance acts on the a-subunit of Na+,K+-ATPase inhibiting the Na+-pump activity. In the vascular smooth muscle this effect leads to intracellular Na+ accumulation that reduces the activity of the Na+/Ca2+ exchanger and to an increased vascular tone. It was also suggested that circulating ouabain, even in the nanomolar range, sensitizes the vascular smooth muscle to vasopressor substances. We tested the latter hypothesis by studying the effects of ouabain in the micromolar and nanomolar range on phenylephrine (PE)-evoked pressor responses. The experiments were performed in normotensive and hypertensive rats in vivo, under anesthesia, and in perfused rat tail vascular beds. The results showed that ouabain pretreatment increased the vasopressor responses to PE in vitro and in vivo. This sensitization after ouabain treatment was also observed in hypertensive animals which presented an enhanced vasopressor response to PE in comparison to normotensive animals. It is suggested that ouabain at nanomolar concentrations can sensitize vascular smooth muscle to vasopressor stimuli possibly contributing to increased tone in hypertension

Effects of small doses of ouabain on the arterial blood pressure of anesthetized hypertensive and normotensive rats

Ouabain increases vascular resistance and may induce hypertension by inhibiting the Na+ pump. The effects of 0.18 and 18 µg/kg, and 1.8 mg/kg ouabain pretreatment on the phenylephrine (PHE; 0.1, 0.25 and 0.5 µg, in bolus)-evoked pressor responses were investigated using anesthetized normotensive (control and uninephrectomized) and hypertensive (1K1C and DOCA-salt treated) rats. Treatment with 18 µg/kg ouabain increased systolic and diastolic blood pressure in all groups studied. However, the magnitude of this increase was larger for the hypertensive 1K1C and DOCA-salt rats than for normotensive animals, while the pressor effect of 0.18 µg/kg ouabain was greater only in DOCA-salt rats. A very large dose (1.8 mg/kg) produced toxic effects on the normotensive control but not on uninephrectomized or 1K1C rats. Rat tail vascular beds were perfused to analyze the effects of 10 nM ouabain on the pressor response to PHE. In all animals, 10 nM ouabain increased the PHE pressor response, but this increase was larger in hypertensive DOCA-salt rats than in normotensive and 1K1C rats. Results suggested that a) increases in diastolic blood pressure induced by 18 µg/kg ouabain were larger in hypertensive than normotensive rats; b) in DOCA-salt rats, smaller ouabain doses had a stronger effect than in other groups; c) hypertensive and uninephrectomized rats were less sensitive to toxic doses of ouabain, and d) after treatment with 10 nM ouabain isolated tail vascular beds from DOCA-salt rats were more sensitive to the pressor effect of PHE than those from normotensive and 1K1C hypertensive rats. These data suggest that very small doses of ouabain, which might produce nanomolar plasma concentrations, enhance pressor reactivity in DOCA-salt hypertensive rats, supporting the idea that endogenous ouabain may contribute to the increase and maintenance of vascular tone in hypertension.

Erythrocytes may contain a ouabain-insensitive K+-ATPase which plays a role in internal K+ balance

Erythrocytes are useful in evaluating K+ transport pathways involved in internal K+ balance. Several forms of H+,K+-ATPase have been described in nephron segments active in K+ transport. Furthermore, the activity of a ouabain-insensitive isoform of H+,K+-ATPase expressed in collecting duct cells may be modulated by acid-base status. Various assays were performed to determine if a ouabain-insensitive K+-ATPase is present in rat erythrocytes and, if so, whether it plays a role in internal K+ balance. Kinetic studies demonstrated that maximal stimulation of enzyme activity was achieved with 2.5 mM K+ at pH 7.4. Subsequent experiments were performed on erythrocyte membranes collected from animals submitted to varying degrees of K+ homeostasis: control rats, K+-depleted rats, K+-loaded rats, and rats rendered hyperkalemic due to acute renal failure. As observed in the collecting duct cell studies, there was a significant decrease in the activity of ouabain-insensitive K+-ATPase in the erythrocytes of both K+-loaded and metabolically alkalotic K+-depleted rats. However, this enzyme activity in erythrocyte membranes of rats with metabolic acidosis-related hyperkalemia was similar to that of control animals. This finding may be interpreted as resulting from two potentially modulating factors: the stimulating effect that metabolic acidosis has on K+-ATPase and the counteracting effect that hyperkalemia and uremia have on metabolic acidosis. In summary, we present evidence of a ouabain-insensitive K+-ATPase in erythrocytes, whose activity is modulated by acid-base status and K+ levels.

A low concentration of ouabain (0.18 µg/kg) enhances hypertension in spontaneously hypertensive rats by inhibiting the Na+ pump and activating the renin-angiotensin system

We investigated the effects of low ouabain concentrations on systolic (SAP) and diastolic (DAP) arterial pressures and on pressor reactivity in 3-month-old male spontaneously hypertensive rats (SHR). Arterial blood pressure (BP) and pressor reactivity to phenylephrine (PHE) were investigated before and after 0.18 μg/kg ouabain administration (N = 6). The influence of hexamethonium (N = 6), canrenone (N = 6), enalapril (N = 6), and losartan (N = 6) on ouabain actions was evaluated. Ouabain increased BP (SAP: 137 ± 5.1 to 150 ± 4.7; DAP: 93.7 ± 7.7 to 116 ± 3.5 mmHg; P<0.05) but did not change PHE pressor reactivity. Hexamethonium reduced basal BP in control but not in ouabain-treated rats. However, hexamethonium + ouabain increased DAP sensitivity to PHE. Canrenone did not affect basal BP but blocked ouabain effects on SAP. However, after canrenone + ouabain administration, DAP pressor reactivity to PHE still increased. Enalapril and losartan reduced BP and abolished SAP and DAP responses to ouabain. Enalapril + ouabain reduced DAP reactivity to PHE, while losartan + ouabain reduced SAP and DAP reactivity to PHE. In conclusion, a small dose of ouabain administered to SHR increased BP without altering PHE pressor reactivity. Although the renin-angiotensin system (RAS), Na+ pump and autonomic reflexes are involved in the effects of ouabain on PHE reactivity, central mechanisms might blunt the actions of ouabain on PHE pressor reactivity. The effect of ouabain on SAP seems to depend on the inhibition of both Na+ pump and RAS, whereas the effect on DAP seems to depend only on RAS.

Hypertensive effects of the iv administration of picomoles of ouabain

Ouabain, an endogenous digitalis compound, has been detected in nanomolar concentrations in the plasma of several mammals and is associated with the development of hypertension. In addition, plasma ouabain is increased in several hypertension models, and the acute or chronic administration of ouabain increases blood pressure in rodents. These results suggest a possible association between ouabain and the genesis or development and maintenance of arterial hypertension. One explanation for this association is that ouabain binds to the α-subunit of the Na+ pump, inhibiting its activity. Inhibition of this pump increases intracellular Na+, which reduces the activity of the sarcolemmal Na+/Ca2+ exchanger and thereby reduces Ca2+ extrusion. Consequently, intracellular Ca2+ increases and is taken up by the sarcoplasmic reticulum, which, upon activation, releases more calcium and increases the vascular smooth muscle tone. In fact, acute treatment with ouabain enhances the vascular reactivity to vasopressor agents, increases the release of norepinephrine from the perivascular adrenergic nerve endings and promotes increases in the activity of endothelial angiotensin-converting enzyme and the local synthesis of angiotensin II in the tail vascular bed. Additionally, the hypertension induced by ouabain has been associated with central mechanisms that increase sympathetic tone, subsequent to the activation of the cerebral renin-angiotensin system. Thus, the association with peripheral mechanisms and central mechanisms, mainly involving the renin-angiotensin system, may contribute to the acute effects of ouabain-induced elevation of arterial blood pressure.

Chronic ouabain treatment exacerbates blood pressure elevation in spontaneously hypertensive rats: the role of vascular mechanisms

Objective Hypertensive rats are more sensitive to the pressor effects of acute ouabain than normotensive rats. We analyzed the effect of chronic ouabain (similar to 8.0 mu g/day, 5 weeks) treatment on the blood pressure of spontaneously hypertensive rats (SHRs) and Wistar-Kyoto rats and the contribution of vascular mechanisms. Methods Responses to acetylcholine and phenylephrine were analyzed in isolated tail arteries. Protein expression of endothelial nitric oxide synthase and cyclooxygenase-2 (COX-2) were also investigated. Results Ouabain treatment enhanced blood pressure only in SHRs. The pD(2) for acetylcholine was decreased in arteries from SHRs compared with Wistar-Kyoto rats, and ouabain did not change this parameter. However, ouabain was able to increase the pD(2) to phenylephrine in SHRs. Nitric oxide synthase inhibition with N(G)-nitro-L-arginine methyl ester or potassium channel blockade by tetraetylamonium increased the response to phenylephrine in SHRs, with a smaller increase in response observed in ouabain-treated SHRs. In addition, indomethacin (a COX inhibitor) and ridogrel (a thromboxane A(2) synthase inhibitor and prostaglandin H(2)/thromboxane A(2) receptor antagonist) decreased contraction to phenylephrine in tail rings from ouabain-treated SHRs. Protein expression of endothelial nitric oxide synthase was unaltered following ouabain treatment in SHRs, whereas COX-2 expression was increased. Conclusion Chronic ouabain treatment further increases the raised blood pressure of SHRs. This appears to involve a vascular mechanism, related to a reduced vasodilator influence of nitric oxide and endothelium-derived hyperpolarizing factor and increased production of vasoconstrictor prostanoids by COX-2. These data suggest that the increased plasma levels of ouabain could play an important role in the maintenance of hypertension and the impairment of endothelial function. J Hypertens 27:1233-1242 (C) 2009 Wolters Kluwer Health vertical bar Lippincott Williams & Wilkins.

Chronic ouabain treatment increases the contribution of nitric oxide to endothelium-dependent relaxation

The aim of this study was to analyze the contribution of nitric oxide, prostacyclin and endothelium-dependent hyperpolarizing factor to endothelium-dependent vasodilation induced by acetylcholine in rat aorta from control and ouabain-induced hypertensive rats. Preincubation with the nitric oxide synthase inhibitor N-omega-nitro-L-arginine methyl esther (L-NAME) inhibited the vasodilator response to acetylcholine in segments from both groups but to a greater extent in segments from ouabain-treated rats. Basal and acetylcholine-induced nitric oxide release were higher in segments from ouabain-treated rats. Preincubation with the prostacyclin synthesis Inhibitor tranylcypromine or with the cyclooxygenase inhibitor indomethacin inhibited the vasodilator response to acetylcholine in aortic segments front both groups. The Ca(2+)-dependent potassium channel blocker charybdotoxin inhibited the vasodilator response to acetylcholine only In segments from control rats. These results indicate that hypertension induced by chronic ouabain treatment is accompanied by increased endothelial nitric oxide participation and impaired endothelium-dependent hyperpolarizing factor contribution In acetylcholine-induced relaxation. These effects might explain the lack of effect of ouabain treatment oil acetylcholine responses in rat aorta.

Long-Term Ouabain Treatment Impairs Vascular Function in Resistance Arteries

Background/Aims: The purpose of this study was to examine the cardiovascular effects of long-term ouabain treatment at different time points. Methods: Systolic blood pressure (SBP) was measured by tail-cuff method in male Wistar rats treated with ouabain (approx. 8.0 mu g.day(-1)) or vehicle for 5, 10 and 20 weeks. Afterwards, vascular function was assessed in mesenteric resistance arteries (MRA) using a wire myograph. ROS production and COX-1 and COX-2, TNF-alpha, and IL-6 protein expression were investigated. Results: SBP was increased by ouabain treatment up to the 6th week and remained stable until the 20th week. However, noradrenaline-induced contraction increased only in MRA in rats treated with ouabain for 20 weeks. NOS inhibition and endothelium removal increased the noradrenaline response, but to a smaller magnitude in MRA in the ouabain group. Moreover, inhibition of COX-2 or incubation with superoxide dismutase restores noradrenaline-induced contraction in the 20-week ouabain group to control levels. ROS production as well as COX-2, IL-6 and TNF-alpha protein expression increased in MRA in this group. Conclusion: Although ouabain treatment induced hypertension in all groups, a larger noradrenaline induced contraction was observed over 20 weeks of treatment. This vascular dysfunction was related to COX-2-derived prostanoids and oxidative stress, increased pro-inflammatory cytokines and reduced NO bioavailability. Copyright (C) 2011 S. Karger AG, Basel

Time-dependent increases in ouabain-sensitive Na(+), K(+)-ATPase activity in aortas from diabetic rats: The role of prostanoids and protein kinase C

Aims: Na(+), K(+)-ATPase activity contributes to the regulation of vascular contractility and it has been suggested that vascular Na(+), K(+)-ATPase activity may be altered during the progression of diabetes; however the mechanisms involved in the altered Na(+), K(+)-ATPase activity changes remain unclear. Thus, the aim of the present study was to evaluate ouabain-sensitive Na(+), K(+)-ATPase activity and the mechanism(s) responsible for any alterations on this activity in aortas from 1- and 4-week streptozotocin-pretreated (50 mg kg(-1), i.v.) rats. Main methods: Aortic rings were used to evaluate the relaxation induced by KCl (1-10 mM) in the presence and absence of ouabain (0.1 mmol/L) as an index of ouabain-sensitive Na(+), K(+)-ATPase activity. Protein expression of COX-2 and p-PKC-beta II in aortas were also investigated. Key findings: Ouabain-sensitive Na(+), K(+)-ATPase activity was unaltered following 1-week of streptozotocin administration, but was increased in the 4-week diabetic aorta (27%). Endothelium removal or nitric oxide synthase inhibition with L-NAME decreased ouabain-sensitive Na(+), K(+)-ATPase activity only in control aortas. In denuded aortic rings, indomethacin. NS-398, ridogrel or Go-6976 normalized ouabain-sensitive Na(+), K(+)-ATPase activity in 4-week diabetic rats. In addition, COX-2 (51%) and p-PKC-beta II (59%) protein expression were increased in 4-week diabetic aortas compared to controls. Significance: In conclusion, diabetes led to a time-dependent increase in ouabain-sensitive Na(+), K(+)-ATPase activity. The main mechanism involved in this activation is the release of TxA(2)/PGH(2) by COX-2 in smooth muscle cells, linked to activation of the PKC pathway. (C) 2010 Elsevier Inc. All rights reserved.

Influence of N-methyl-D-aspartate receptors on ouabain activation of nuclear factor-kappa B in the rat hippocampus

It has been shown that ouabain (OUA) can activate the Na,K-ATPase complex and mediate intracellular signaling in the central nervous system (CNS). Inflammatory stimulus increases glutamatergic transmission, especially at N-methyl-D-aspartate (NMDA) receptors, which are usually coupled to the activation of nitric oxide synthase (NOS). Nuclear factor-kappa B (NF-kappa B) activation modulates the expression of genes involved in development, plasticity, and inflammation. The present work investigated the effects of OUA on NF-kappa B binding activity in rat hippocampus and the influence of this OUA-Na,K-ATPase signaling cascade in NMDA-mediated NF-kappa B activation. The findings presented here are the first report indicating that intrahippocampal administration of OUA, in a concentration that did not alter Na,K-ATPase or NOS activity, induced an activation of NF-kappa B, leading to increases in brain-derived neurotrophic factor (Bdnf), inducible NOS (iNos), tumor necrosis factor-alpha (Tnf-alpha), and B-cell leukemia/lymphoma 2 (Bcl2) mRNA levels. This response was not linked to any significant signs of neurodegeneration as showed via Fluoro-Jade B and Nissl stain. Intrahippocampal administration of NMDA induced NF alpha B activation and increased NOS and alpha 2/3-Na,K-ATPase activities. NMDA treatment further increased OUA-induced NF-kappa B activation, which was partially blocked by MK-801, an antagonist of NMDA receptor. These results suggest that OUA-induced NF-kappa B activation is at least in part dependent on Na,K-ATPase modulatory action of NMDA receptor in hippocampus. The interaction of these signaling pathways could be associated with biological mechanisms that may underlie the basal homeostatic state linked to the inflammatory signaling cascade in the brain. (c) 2011 Wiley Periodicals, Inc.